Effects of Phosphorylation by CAK on Cyclin Binding by CDC2 and CDK2

作者：Dipty Desai, Holly C. Wessling, Robert P. Fisher, David O. Morgan · 发表于：Molecular and Cellular Biology · 年份：1995 · DOI：10.1128/mcb.15.1.345 · 被引用次数：148 · 研究领域：Cancer-related Molecular Pathways、Microtubule and mitosis dynamics、Ubiquitin and proteasome pathways

The cyclin-dependent protein kinases (CDKs) are activated by association with cyclins and by phosphorylation at a conserved threonine residue by the CDK-activating kinase (CAK). We have studied the binding of various human CDK and cyclin subunits in vitro, using purified proteins derived from baculovirus-infected insect cells. We find that most CDK-cyclin complexes known to exist in human cells (CDC2-cyclin B, CDK2-cyclin A, and CDK2-cyclin E) form with high affinity in the absence of phosphorylation or other cellular components. One complex (CDC2-cyclin A) forms with high affinity only after CAK-mediated phosphorylation of CDC2 at the activating threonine residue. CDC2 does not bind with high affinity to cyclin E in vitro, even after phosphorylation of the CDC2 subunit. Thus, phosphorylation is of varying importance in the formation of high-affinity CDK-cyclin complexes.